Various procedures are known in the biomedical art employing biologically active compounds containing a detectable signal-generating dye moiety which may, for example, be a moiety which emits electromagnetic radiation, e.g. a fluorescent, chemiluminescent or bioluminescent substance. The biologically active compound may, for example, be a DNA probe, e.g. a labeled DNA probe of the type used in detecting complimentary DNA sequences; an enzyme; enzyme inhibitor; antigen; antibody; hapten, etc.
In recent years, much attention has been focused upon labeled-reagent immunoassays for the detection of body fluid antigens, hormones, infectious agents, serum antibodies, and the like. Consequently, the patent literature is replete with disclosures of various assays involving a labeled-reagent reaction between antigens and antibodies to provide a detectable signal, e.g. a change in color, emission of electromagnetic radiation, etc. These assays can be said to involve an immunological interaction between a ligand and an antiligand, wherein at least one of the two reactants contains a substance or a precursor of a substance which can produce the detectable signal as a function of the immunological ligand-antiligand interaction.
One class of labels commonly used in such assays are fluorescent dyes or fluorophors. Both heterogeneous and homogeneous specific binding assays employing fluorescent labeled conjugates are well known and are reported in the patent literature. By way of illustrating the general state of the art with respect to the use of fluorescers as labels in specific binding assays, mention may be made of U.S. Pat. Nos. 3,992,631; 3,999,948; 4,020,151; 4,025,310; 4,036,946; 4,058,732; 4,115,699; 4,220,450; and 4,238,195.
In general, it can be said that fluorescent labels for use in these assays should exhibit the highest possible fluorescent efficiency, have a relatively long emission wavelength (above 500 nm), and be capable of being bound covalently to the liquid or antiligand without negatively affecting the conjugation properties.
In addition, the fluorescent labels should ideally have a high Stokes shift (energy difference between absorption and emission). A high Stokes shift is desirable as it reduces interference by scattered light and system fluorescent background. Assay systems relying upon energy transfer, e.g., from a chemiluminescent source to a fluorescent label to provide a detectable signal are described, for example, in the aforementioned U.S. Pat. No. 4,220,450.
The present invention relates to novel fluorescent conjugates and their use in biological diagnostic elements.
It is therefore an object of the invention to provide novel fluorescent compounds.
It is another object to provide novel fluorescent compounds which include a biologically active moiety and a dye moiety.
It is a further object to provide novel fluorescent compounds which include a substantially achromophoric moiety.
Still another object is to provide biological diagnostic elements which utilize such novel fluorescent compounds.